Regulation of fibrinolytic activity by localization of inhibitors to fibrin(ogen)

Abstract Fibrin plays a key role in fibrinolysis, acting not only as a substrate but also as a regulator of activity. As well as stimulating plasminogen activation, it controls interactions between proteases and inhibitors, both protecting proteases from inhibition and, conversely, localizing inhibitors. The principal inhibitor of plasmin, α2-antiplasmin, is cross-linked to fibrinogen and fibrin, inhibiting fibrinolysis. Our studies have shown that a second inhibitor, PAI-2, is also cross-linked to fibrinogen and fibrin, by either factor XIIIa or tissue transglutaminase. These inhibitors are both members of the serpin family but the cross-linking sites are quite unrelated. Cross-links are formed between glutamine residues in the inhibitors and lysine residues in fibrin(ogen). The Gln residues involved are at position 2 in the N-terminus of α2-AP and at position 83 and 86 in PAI-2, located in a loop between helices C and D. All cross-linking observed was to the Aα chain of fibrin(ogen). The two inhibitors did not compete for cross-linking sites. α2-AP binds only to Lys 303 of the Aα chain and a 30-residue peptide based on the sequence around this Lys competed with fibrinogen for cross-linking to α2-AP but not for cross-linking to PAI-2. PAI-2 was cross-linked to several Lys residues (but not Lys 303) in the Aα chain, as shown by tryptic digestion and mass spectrometry. PAI-2 was cross-linked to Lys 148, 176, 183 and 467 by tissue transglutaminase and to Lys 148, 176, 230 and 413 by factor XIIIa. The activity of PAI-2 was not affected by cross-linking, so that this is a mechanism whereby it can be covalently bound to fibrinogen and retained in a fibrin clot, without loss of activity towards u-PA and two-chain t-PA. PAI-1, the other major inhibitor of fibrinolysis, also binds to fibrin but we find no evidence for its being cross-linked. All three inhibitors achieve high local concentrations on fibrin, which they protect from lysis by t-PA, u-PA and plasmin. The inhibitors differ in their major sources in blood, with α2-AP present at high concentrations in plasma, PAI-1 primarily in platelets, and PAI-2 a product of stimulated monocytes, giving them distinct and complementary roles in stabilizing fibrin in different physiological and pathological locations.